A. Botulinum Toxin: Mechanism of Action
Botulinum neurotoxin is a toxin isolated from a strain of Clostridium botulinum, that acts at the neuromuscular junction by inhibiting release of acetylcholine. Botulinum toxin is initially formed as a single-chain polypeptide that is cleaved to form a light chain that is bound to a heavy chain through a disulfide bond. The denervating effect of botulinum toxin occurs through: 1) the binding of the heavy chain to high-affinity receptors at the presynaptic terminal; 2) internalization of botulinum toxin through endocytosis; 3) translocation of the light chain into the cytoplasm of the nerve terminal; and 4) the endo-metalloprotease activity of the light chain (zinc cofactor) cleaves specific synaptic proteins that inhibit fusion of synaptic vesicles with the presynaptic membrane, thereby inhibiting the release of acetylcholine contained in the vesicles. Absent acetylcholine, the muscle does not receive the necessary signal for the muscle to contract. Subsequent to injection, neurogenic muscular atrophy ensues after several weeks.
B. Botulinum Toxin: Clinical Applications
A deadly toxin at high concentrations and quantities, botulinum toxin has been used as a valuable therapeutic for the treatment of many neuromuscular diseases (e.g., dystonia, hemifacial spasm, bruxism, spasticity, cerebral palsy, torticollis), as well as sensory disorders and cutaneous disorders (myofascial pain, migraine, tension headaches, neuropathy, hyperhydrosis), and in the treatment of disorders involving inflammation. The therapeutic value of botulinum toxin in its ability to produce local regional denervation of specific muscles and tissues.
The action of botulinum toxin on nerve terminals is irreversible. Axon sprouting, however, reverses the denervating effects of the toxin within two to six months. Consequently, a variety of conditions and disorders require repeated administration of the neurotoxin. Resistance to botulinum toxin is an important clinical consequence and problem resulting from repeated administration of botulinum toxin and the production of neutralizing antibodies. (Naumann et al. (1998) J. Neurol. Neurosurg. Psychiatry 65: 924-927; Hauna et al. (1998) J. Neurol. Neurosurg. Psychiatry 66: 612-616). The problem is most noted in high-dose applications such as cervical dystonia, however, immunity and resistance to the botulinum neurotoxin may occur with lower dose applications such as blepharospasm. Recently, the inventor observed that resistance can occur even with low-dose cosmetic applications, such as the treatment of facial rhytides. Accordingly, it is an object of the present invention to provide high-potency formulations and corresponding methods that reduce the likelihood of neutralizing antibodies in subjects treated with botulinum toxin.
The antigenicity of botulinum toxin stimulates antibody formation that reduces and most often completely obliterates the therapeutic effectiveness of botulinum-neurotoxin-based pharmaceuticals and may ultimately lead to abandonment of botulinum therapy. Several strategies to minimize the development of resistance have been directed toward reducing the antigenicity of the botulinum neurotoxin itself. For example, pegylated botulinum toxin (botulinum toxin covalently coupled to polyethylene glycol) have been developed for the treatment of neuromuscular disorders. Pegylation of the toxin is site directed thereby reducing antigenicity without interfering with neurotoxic effect. (See, U.S. Patent Publication No. 20020197278). Also, hybrid-toxin molecules with reduced antigenicity have been synthesized using the targeting and internalization portion (heavy chain) of one toxin serotype and the catalytic portion of a different serotype (light chain). The hybrid-toxin molecules exhibit reduced antigenicity but retain the inherent-binding specificity of the botulinal-heavy chain from the first serotype and the catalytic potency of the light chain from the second serotype. (See, U.S. Pat. No. 6,444,209).
Reduced antigenicity may also be achieved by further purifying the neurotoxin by reducing the antigenic complex proteins and other clostridial proteins associated with the toxin. (See, U.S. Pat. Nos. 5,756,468 and 5,512,547). Type A neurotoxin produced by C. botulinum is present as part of a complex of at least seven different noncovalently bound proteins. These nontoxic proteins range in size from about 17 to 118 kD and are associated with the neurotoxin that has a molecular weight of about 147 kD. (Goodnough et al. (1993) Appl. Environ. Microbiol. 59: 2339-2342; Gimenez et al. (1993) Protein Chem. 12: 349-361; DasGupta (1980) Canad. J. Microbiol. 26: 992-997). Some of the non-toxic proteins associated with the various toxin complexes have hemagglutinating abilities (Sugiyama (1980) Microbiol. Rev. 44: 419-448; Somers et al. (1991) J. Protein Chem. 10:415-425). In particular, non-neurotoxic fractions of the L complexes of type A, B, C, and D have been shown to have hemagglutinating activity. Hemagglutinin fractions isolated from the different serotypes show some serological cross-reactivity. Non-toxic fractions from type A and B serotypes cross-react (Goodnough and Johnson (1993) Appl. Environ. Microbiol. 59: 2339-2342) as do non-toxic fractions from types E and F. The non-toxic fractions of types C1 and D are antigenically identical as determined by Ouchterlony diffusion (Sakaguchi et al. (1974) Jpn. J. Med. Sci. Biol. 27: 161-170). By removing these proteins, more neurotoxin may be delivered to a therapeutic site with less antigenic proteins that may lead to the production of neutralizing antibodies.
C. Complications Associated with Conventional Botulinum-Toxin Formulations
Substantial differences in the complication rate have also been noted at therapeutic quantities of different botulinum preparations. Side effects such as those resulting from diffusion of the botulinum toxin from the site of administration appear to be dependent on the formulation of botulinum toxin. For instance, dysphagia rates (difficulty swallowing) is a well-known complication of botulinum toxin administration when used for the treatment of cervical dystonia. (Borodic et al. (1990) Botulinum A toxin for the treatment of spasmodic torticollis. Dysphagia and Regional Toxin Spread. Head & Neck, 12: 392-398; incorporated herein by reference in its entirety). Differences in the rate of this complication between various formulations has been well appreciated when reviewing prior art literature between 1984-1995. Furthermore differences in the rate of ptosis (drooping eyelid) have been reported when comparing various immunotypes and different preparations of the same immunotype (see Table 1). It has become well accepted that this complication is the result of diffusion of botulinum toxin away from the injections sites, a property which is in conflict with the clinical goal of containing the denervating or biologic effect to a specific target region. The formulations and methods disclosed herein contain the biologic effect of the neurotoxin to a targeted anatomic region and thereby reduce the diffusion potential of the botulinum toxin pharmaceutical and decrease the associated side effects.
TABLE 2Diffusion-related complications between various pharmaceuticalformulations of botulinum toxin.ComplicationBOTOX ®DYSPORT ®2MYOBLOC ®3Ptosis1<2%12-15%30-40%Dysphagia<2%14-21%10-17%1Nussgens et al. (1997) Comparison of two botulinum-toxin preparations in the treatment of essential blepharospasm. Graefes Arch Clin Exp Ophthalmol 235(4): 197-199.2Phase 3 Studies 1998-1989 for Oculinum Meta-analysis of clinical studies on Dysphagia and Botulinum 1995 at NIH (Borodic).3Lew et al. (1997) Botulinum toxin type B: a double-blind, placebo-controlled, safety and efficacy study in cervical dystonia. Neurology 49(3): 701-707.
In 1991, Borodic et al. developed a histologic model demonstrating a histochemical and morphologic diffusion gradient from point injections of botulinum toxin. (Borodic et al. (1991) Botulinum toxin: Clinical and scientific aspects. Opthamology Clinics of North America 4: 491-503; incorporated herein by reference in its entirety). The gradient was dose dependent over single muscle strips and capable of crossing fascial planes. The diffusion model was further demonstrated on the facial wrinkling pattern of the human forehead. (Borodic et al (1992) Botulinum toxin for spasmodic torticollis, multiple vs single point injections per muscle. Head and Neck 14: 33-37). Diffusion was thereafter used to explain the mechanism for dysphagia after surface injections of botulinum injection for the human neck and ptosis (drooping eyelid) after periocular injections for the treatment of essential blepharospasm. Ptosis results from diffusion of neuromuscular blocking activity from the lid edge to the muscular portion of the upper eyelid retractor, which lies in the upper orbital space. Dysphagia results from diffusion of neuromuscular weakening effect from the sternomastoid muscle, targeted for treatment of torticollis, to the peripharygeal musculature which generates the force for effective swallowing. From both histologic models and clinical experience, diffusion appears directly related to the quantity of toxin (in LD50 units) administered. Consequently, the greater the quantity of toxin used as an injection in units used, the greater the diffusion from that point. A review of the scientific literature from the 1980's and early 1990's reveals that dysphagia is more commonly with observed with DYSPORT® than BOTOX®. Recently, from studies done at European centers, the differences in dysphagia rates have been confirmed (Ranoux et al. (2002) Respective potencies of DYSPORT® and BOTOX®: a double blind, randomized, crossover study in cervical dystonia. J. Neurol. Neurosurg. Psychiatry 72: 459-462). Differences in ptosis rates for the treatment of blepharospasm have also been observed comparing BOTOX®. Ptosis is less frequently observed with BOTOX° (Nussgens et al. (1997) Comparison of two botulinum-toxin preparations in the treatment of essential Blepharospasm. Graefes Arch Clin Exp Opthalmol. 235(4): 197-199). Major differences in the ptosis complication have also been reported when using botulinum toxin type B for the treatment of glabellar and forehead wrinkles when compared to botulinum type A (BOTOX®). (Holck et al. Comparison of High Dose Botulinum Toxin Type B to Botulinum Type A in the Treatment of Lateral Canthal Rhytides American Society of Ophthalmic Plastic and reconstructive Surgeons Annual Meeting, Anaheim, Calif. 11-14-03).
Prior to this invention, the in vivo binding of sequestration agents, such as albumin, to botulinum toxin has never been identified as important to clinical effectiveness of botulinum-toxin-based pharmaceuticals. By enhancing regional sequestration of the neurotoxin and facilitating saturation of neurotoxin receptors on neural tissues, high-concentration-albumin formulations improve the clinical effectiveness of botulinum toxin and reduce side effects such as those resulting from diffusion of the botulinum toxin from the site of administration. There has been no prior suggestion that increasing the albumin concentration, for example, relative to the neurotoxin, could enhance the effectiveness for the treatment of human disease. The existing botulinum toxin preparations currently available for clinical practice are BOTOX®, DYSPORT®, and MYOBLOC®. The present invention identifies the mechanism and provides compositions of improved utility of botulinum-toxin-based pharmaceuticals by increasing the concentration of a sequestration agent and other viscous agents to enhance sequestration and improve the effectiveness where other available botulinum toxin preparations have failed.
D. Sequestration
Albumin was initially used to formulate botulinum-toxin-based pharmaceuticals because of its stabilizing effect on the biologic activity of the neurotoxin at high dilutions (see Schantz, Botulinum Toxin Therapy, Marcel Dekker 1994). Dilution of the purified botulinum toxin crystals with physiologic saline or water would cause the biologic activity and pharmaceutical properties to be lost at high dilutions. Additionally, the albumin has been reported to help keep the neurotoxin molecule from binding to glass containers. During the pre-clinical development of BOTOX® or any other botulinum toxin prepared for pharmaceutical use, there was no appreciation for the importance of albumin in the formulation other than a dilution stabilizer and excipient to keep the neurotoxin from binding to glass.
BOTOX® and DYSPORT° are derived from different strains of Clostridial species. BOTOX' is derived from the Hall strain of Clostridium botulinum originally maintained by the University of Wisconsin, whereas DYSPORT® is derived from British Microbiology Collection. Immunologic cross reactivity exists between the products as both products were derived from immunotype A strains. Despite similar immunotypes, the clinical responses between BOTOX® and DYSPORT® may be explained by the differences in the excipients used in each formulation. The difference in human serum albumin concentrations between BOTOX® and DYSPORT® are outlined in Table 3.
TABLE 3Human Serum Albumin content of variouspharmaceutical formulations of botulinum toxin.FormulationAlbumin1LD50/μg albuminBOTOX ®500 μg0.2DYSPORT ®125 μg5.01Albumin is represented in mg per 100 LD50 units of botulinum toxin. Other differences exist including the presence of stabilizing sugars, Lactose is used in DYSPORT ® and not used in BOTOX ®.
The albumin discrepancy between BOTOX® and DYSPORT® is almost identical to the difference in dose requirements observed between BOTOX® and DYSPORT® in multiple clinical studies. The correlation between the albumin ratio/clinical potency ratio is further strengthened by changes in pharmacologic properties of DYSPORT® when albumin is added to the vials using a mouse hemidiaphram animal model. Wohlfahrt et al. noted using this model that adding albumin to one vials of DYSPORT® brought biologic activity higher using the mouse hemi-diaphragm model. (Biglalke et al (2001) Botulinum A toxin: DYSPORT® improvement of biological availability. Exp. Neurol. 168(1): 162-170). The authors suggested the increased biologic activity resulted from increased stability as measured with the mouse LD50 bioassay afforded by the albumin concentration increase. (Biglalke et al (2001) Botulinum A toxin: DYSPORT® improvement of biological availability. Exp. Neurol. 168(1): 162-170). The authors explained the differences of albumin on the LD50 bioassay without reference to mechanism of action in tissues or pharmacologic-pharmacokinetic importance, that is, in vivo albumin binding, enhanced sequestration, and improvement in therapeutic effects. The same authors further observed in a rat-diaphragm preparation, that the addition of albumin to the BOTOX® preparation could not substantially increase regional denervative effects and did not advocate any changes in formulation. The findings of these researchers concluded that there was an effect of the albumin concentration on the LD50 measurements, however, their work did not demonstrate any increased potency of BOTOX® on regional denervation or that DYSPORT® could be enhanced to give any greater denervation potency over BOTOX®. Their work was limited by the in vitro nature of their experiments, that is, using a non-blood-perfused-animal dissection of a motor nerve (phrenic nerve) and diaphragm muscle, which fails to accounts for dilutions and tissue fluid flow capable of washing injected toxin away from targeted tissue prior to binding with the nerve axon terminal receptors. The real time application requires an in vivo analysis of the effects of albumin on regional denervation as outlined in the following experiments. Their work did identify reasons for differences in LD50 as measured by the mouse lethality assay. These workers, however, concluded that no improvements in potency or effectiveness could be made over existing BOTOX® preparation. (Hanover Germany International Botulinum Toxin Meeting 2002).
Differences in potency, issues relating diffusion and containment of the biologic effect, and the development of resistance are important in the pharmacology of botulinum-based pharmaceuticals. Described herein is a method for altering compositions of botulinum based pharmaceuticals to enhance potency, increase sequestration of the botulinum toxin and limit adverse effects of botulinum-based pharmaceuticals.